One of the main problems, which currently limit the application of gene therapy and genetic vaccines into common clinical practice, is the insufficient transporting efficiency of nucleic acid (NA) fragments through the cell wall and their following internalization into the cell nucleus, the so-called transfection (Miller, A. D. 2004; Zhang, S. B. et al. 2010). In this case, the drug is a fragment of negatively charged nucleic acid, which must be able to penetrate through the cell and nuclear membranes. It is essentially a gene correction by insertion of (a) correcting gene(s) and reduction of activity of unsuitable genes. DNA vaccines represent a specific area of gene therapy, in which the target cells are antigen presenting cells of the immune system (especially dendritic cells and monocytes; Saha, R. et al. 2011).
The penetration of the negatively charged NA fragment through the phospholipid bilayer of the cell wall plays a key role in this process. In the last decade, this fact has initiated a broad and intensive research focused on the development of carriers (vectors) that would be able to effectively transport the NA through the cell membrane. These vectors must also guarantee the protection of the NA from its degradation in vivo (Kirby, A. J. et al. 2003; Miller, A. D. 1998; Zhang, S. B. et al. 2010). The problem of cell membrane transport efficiency occurs also in the case of nucleotide and oligonucleotide antineoplastics and antivirotics (Holý, A. 2003).
The methods for transport of the NA fragment into the target cells use either viral or nonviral vectors. Today, the viral vectors represent the most effective transfection system. Unfortunately, due to possible biological risks (especially unpredictable immune reactions), the introduction of viral vectors into a regular clinical practice is very problematic (Miller, A. D. 1992; Zhang, S. B. et al. 2010). The nonviral vectors can be divided into physical and chemical vectors, according to the method of transporting the NA into the intracellular space. The physical vectors use physical or mechanical disruption of the cell membrane. This enables the insertion of the NA into the intracellular space (Andre, F. M. et al. 2010). The chemical vectors are based on polycationic polymers or on supramolecular self-assembling lipidic systems, which form a complex (polyplex, resp. lipoplex) with the negatively charged NA, wherein the complex can pass through the cell membrane and also protects the NA from the degradation in bloodstream. The most often used cationic polymers are DEAE-dextran (Ohtani, K. et al. 1989), chitosan (Hejazi, R. et al. 2003; Koping-Hoggard, M. et al. 2001), polylysine (Lemaitre, M. et al. 1987), polyethylenimine (Boussif, O. a spol. 1995), and polyamine dendrimers, respectively (Haensler, J. et al. 1993).
At present, polycationic self-assembling lipidic systems (polycationic liposomes) seem to be promising candidates for NA carriers, applicable in human medicine. They are most often formed by synthetic lipopolyamine (so-called cytofectine) and a neutral colipid. Polycationic liposomes, in contrast to viral vectors, are composed from structurally defined molecules and therefore their physical and biological properties can be modulated by structural changes with the aim to increase the transfection ability and to suppress their toxicity. This fact initiated an extensive research in the area of polycationic lipids. Many cationic lipids differing in the character of cationic and hydrophobic domains were prepared (Niculescu-Duvaz, D. et al. 2003; Zhi, D. F. et al. 2010). Many of these cationic lipids are now commercially available as transfection agents and several liposomal formulations were used in clinical tests in gene therapy of cancer and other genetic diseases (Behr, J. P. 1994).
From the structural point of view, the cationic domains represent cationic lipids, the domain of which is composed of polyamines derived from natural spermine or spermidine. They form the most successful class of cationic lipids. Their activity is due to an effective neutralization, precipitation and encapsulation of DNA, and their endosomal and buffering properties (Stewart, L. et al. 2001). Other often occurring cationic domains are, e.g., quaternary ammonium ions, guanidinium motive, nitrogen containing heterocycles, basic amino acids, and short peptides derived therefrom (Niculescu-Duvaz, D. et al. 2003). Hydrophobic domains usually contain one or more aliphatic chains (saturated, unsaturated or fluorinated), or a steroid residue.
The overall geometry of the cationic lipid, i.e. the ratio of the polar and the nonpolar part of the molecule, has a fundamental influence on the formation of structural phases in solution and on the transfection activity. Two-chain lipids, in comparison with the single-chain or three- or multiple-chain lipids, more easily form lipidic bilayers, which close itself into spherical liposomes in water solution. On the other hand, cationic lipids containing one or three aliphatic chains have an increased tendency to form micelles or reverse micells and therefore they show a lower transfection activity and often an increased toxicity (Niculescu-Duvaz, D. et al. 2003; Tsukamoto, M. et al. 1995). Therefore, an overwhelming majority of commonly used cationic lipids contains two aliphatic chains. The most commonly used branching domain is glycerol, which, like in natural amphiphiles, serves for the presentation of two hydrophobic chains (Zuhorn, I. S. et al. 2002). Cationic lipids having this structure can be symmetrical or asymmetrical, depending on the place where the hydrophobic domain is bound. Synthetically easily obtainable secondary amides of amino acids represent an interesting branching principle enabling presentation of two hydrophobic chains (Behr, J. P. et al. 1989). Other structural motives, suitable for multiple presentation of hydrophobic domains, are, e.g., substituted aromatic rings and short peptides (for review see ref. Niculescu-Duvaz, D. et al. 2003). A specific group of hydrophobic domains are sterols with planar structure. Lipids containing a steroidal unit have the tendency to strengthen the lipidic bilayer (Regelin, A. E. et al. 2000). They are mostly derivatives of cholesterol with polycationic domain of polyamine type which are bound in position C(3) via urethane group. Into this category belongs also the commercially available C-DAN (Gao, X. et al. 1991; Keller, M. et al. 2003; Petukhov, I. A. et al. 2010). A broader application of these cationic lipids is limited by the restrained stability of their urethane connecting group. Transfection systems can be based also on amides of cholic acids (Fujiwara, T. et al. 2000).